Project Summary/Abstract Shigella flexneri uses its type three secretion system (T3SS) as a conduit through which effector proteins are shuttled from bacterial to host cell cytoplasm. Once injected, they subvert host functions, promote infection, and defend against host immune responses. While T3SSs have been studied for nearly three decades, details of how T3SS activity is controlled remain largely unclear. We have recently identified the Shigella protein Spa47 as an ATPase whose activity is required for formation of the needle-like type three secretion apparatus, protein effector secretion through the apparatus, and overall Shigella virulence. The absolute reliance of the Shigella T3SS on Spa47 activity suggests it is key in regulating T3SS activity and ultimately pathogen virulence. As a result, we have studied the mechanism of Spa47 catalyzed ATP hydrolysis, finding that a complete Spa47 active site resides at the interface of adjacent Spa47 protomers within the Spa47 homo-oligomer, showing that controlling Spa47 oligomerization is an effective means to regulate ATPase activity and T3SS function. Interaction with the T3SS protein, MxiN, exploits this reliance on Spa47 oligomerization by disrupting Spa47 oligomers and forming a ?chaperoned? MxiN2Spa47 complex that we believe minimizes wasteful ATP hydrolysis and poises the heterotrimer for insertion into the base of the type three secretion apparatus, activating Spa47 and protein secretion. Recent follow-up studies have additionally identified a natural C-terminal variant of the Shigella protein, Spa33, as a potent differential regulator of both Spa47 and MxiN2Spa47 activity, implicating Spa33 as an additional key regulatory component of the Shigella T3SS that likely functions in concert with MxiN to control Spa47 activity in vivo. We have additionally characterized the effects of both post-translational modification of Spa47 and small molecule Spa47 inhibition on in vitro Spa47 activity and in vivo Shigella T3SS function and virulence. Inhibiting Spa47 activity by either method directly correlates to reduced T3SS activity, providing new insight and research directions into intrinsic and extrinsic means of controlling Shigella virulence. To better understand the regulatory role of Spa47 and associated T3SS proteins in Shigella infection, the specific aims of this study are to: 1) Identify and characterize Shigella T3SS protein interactions responsible for Spa47 ATPase regulation. and 2) Dissect T3SS inhibition by both post-translational tyrosine phosphorylation of Spa47 and treatment with small molecule ATPase inhibitors. The proposed studies will determine the influence of Spa33 interaction on activation and regulation of Spa47 and the previously described MxiN2Spa47 complex, determine the environmental triggers and inhibition mechanism behind tyrosine phosphorylation of Spa47, and characterize a series of predicted Spa47 inhibitors already identified by a >7million compound in silico screen. These findings will close a significant gap in the understanding of how an important class of human pathogens control their virulence through appropriate timing of T3SS activation and provide a foundation for exploring non-antibiotic small molecule T3SS inhibitors.